Fine particle measuring apparatus

ABSTRACT

A fine particle measuring apparatus is provided. The fine particle measuring apparatus includes a detection unit configured to detect light emitted from a fine particle and a processing unit having a memory device storing instructions which when executed by the processing unit, cause the processing unit to calculate a corrected intensity value of the detected light and generate spectrum data based on the corrected intensity value.

CROSS REFERENCES TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.13/605,256, filed Sep. 6, 2012, which claims priority to JapanesePriority Patent Application JP 2011-199892 filed in the Japan PatentOffice on Sep. 13, 2011, the entire content of which is herebyincorporated by reference.

BACKGROUND

The present application relates to a fine particle measuring apparatuswhich optically measures a property of a fine particle such as a cell.

A flow cytometer is an apparatus which irradiates fine particles such ascells and beads which flow in a flow cell with light and detectsfluorescence or scattering light emitted from the fine particles so asto optically measure a property of each of the fine particles.

In a case of detecting fluorescence of a cell, for example, cells whichare marked by fluorescence coloring matters are irradiated withexcitation light such as laser light which has appropriate wavelengthand intensity. Then, fluorescence emitted from the fluorescence coloringmatters is condensed by a lens or the like and light in an appropriatewavelength band is selected with a wavelength selecting element such asa filter and a dichroic mirror so as to detect the selected light with alight-receiving element such as a photo multiplier tube (PMT). At thistime, fluorescence from a plurality of fluorescence coloring mattersmarked on cells can be simultaneously detected and analyzed by combininga plurality of wavelength selecting elements and light-receivingelements. Further, the number of fluorescence coloring matters which canbe analyzed can be increased by combining excitation light of aplurality of wavelengths.

As a method for detecting fluorescence with a flow cytometer, there is amethod for measuring intensities of light beams in continuous wavelengthbands as a fluorescence spectrum as well as the method in which aplurality of light beams in discontinuous wavelength bands are selectedwith a wavelength selecting element such as a filter so as to measureintensities of the light beams in respective wavelength bands. Aspectrum type flow cytometer which is capable of measuring afluorescence spectrum divides fluorescence emitted from fine particleswith a spectral element such as a prism and a grating. Then, the flowcytometer detects the divided fluorescence with a light-receivingelement array in which a plurality of light-receiving elements ofdifferent detection wavelength bands are arranged. As thelight-receiving element array, a PMT array or a photodiode array inwhich light-receiving elements which are PMTs or photodiodes areone-dimensionally arranged, or an array in which a plurality ofindependent detection channels such as two-dimensional light-receivingelements which are CCDs or CMOSs are arranged is used.

Japanese Unexamined Patent Application Publication No. 2003-83894 is anexample of related art.

SUMMARY

A measurement value of a flow cytometer includes an error caused byvarious factors. As a correcting method of a measurement error, a methodusing a standard sample of which a fluorescence property is previouslyidentified, for example, is generally used. In this method, arelationship between an output value of current or the like and afluorescence intensity (calibration information) about eachlight-receiving element is acquired based on a reference value obtainedby measuring a plurality of standard samples and calibration isperformed based on this relationship to obtain a measurement value.

In the above-described correcting method, a standard sample has to bemeasured in every measurement by a flow cytometer so as to acquirecalibration information corresponding to an output of laser light and asetting value of a light-receiving element (for example, a voltage orthe like in a case of a PMT). Thus, the above-described method is verycomplicated.

It is desirable to provide a fine particle measuring apparatus which cancorrect a measurement error by simple processing.

According to an embodiment, a fine particle measuring apparatus isprovided that includes a detection unit configured to detect lightemitted from a fine particle, and a processing unit having a memorydevice storing instructions which when executed by the processing unit,cause the processing unit to (a) calculate a corrected intensity valueof the detected light; and (b) generate spectrum data based on thecorrected intensity value.

By correcting an intensity value, which may be obtained in each of aplurality of light-receiving elements by a detection wavelength width ofa corresponding light-receiving element, a measurement error caused bynonlinearity of an optical system of the apparatus can be compensated.

This fine particle measuring apparatus may be a spectrum type fineparticle measuring apparatus which includes a spectral elementconfigured to divide light from the fine particle and a light-receivingelement array in which a plurality of light-receiving elements ofdifferent detection wavelength bands are arranged, as the detectionunit. Especially, the fine particle measuring apparatus may be aspectrum type flow cytometer.

In this fine particle measuring apparatus, it is preferable that theprocessing unit correct the first corrected intensity value by usingsensitivity data of each of the light-receiving elements so as tocalculate a second corrected intensity value. By correcting the firstcorrected value by a relative sensitivity of each of the light-receivingelements, a measurement error caused by sensitivity difference among thelight-receiving elements can be compensated.

Further, the processing unit may form a spectrum chart in which an axisexpresses the detection wavelength and another axis expresses the firstcorrected intensity value or the second corrected intensity value, so asto output the spectrum chart to a display unit. Furthermore, it ispreferable that the processing unit generate spectrum data using thedetection wavelength as a parameter and the first corrected intensityvalue or the second corrected intensity value as another parameter, andcompare the spectrum data with reference spectrum data that is stored ina storage unit, so as to output whether the both data are accorded witheach other or are not accorded with each other to the display unit.

In the embodiment of the present application, “fine particles” widelyinclude physiologically-related fine particles such as cells,microorganisms, and liposome, synthetic particles such as latexparticles, gel particles, and industrial particles, and the like.

The physiologically-related fine particles include a chromosome,liposome, a mitochondrion, organelle, and the like which constitutevarious cells. Cells include animal cells (blood cells and the like) andplant cells. Microorganisms include bacterium such as a coli bacterium,viruses such as a tobacco mosaic virus, fungi such as a yeast cell, andthe like. The physiologically-related fine particles may also include aphysiologically-related polymer such as nucleic acid, protein, and acomplex of nucleic acid and protein. Industrial particles may be organicpolymeric materials, inorganic polymeric materials, metallic materials,or the like. Organic polymeric materials include polystyrene,styrene-divinylbenzen, polymethyl methacrylate, and the like. Inorganicpolymeric materials include glass, silica, magnetic materials, and thelike. Metallic materials include gold colloid, aluminum, and the like.These fine particles commonly have a spherical shape but may have anon-spherical shape. In addition, a size, mass, and the like of thesefine particles are not especially limited.

According to the embodiment of the present application, a fine particlemeasuring apparatus which can correct a measurement error withoutmeasuring a standard sample in each sample analysis and can obtain anaccurate analysis result is provided.

Additional features and advantages are described herein, and will beapparent from the following Detailed Description and the figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a block diagram illustrating the functional configuration of afine particle measuring apparatus according to an embodiment of thepresent application;

FIG. 2 is a schematic diagram illustrating the configuration of adetection unit of the fine particle measuring apparatus;

FIG. 3 is a schematic diagram illustrating the configuration of adetection unit of a fine particle measuring apparatus according toanother embodiment of the present application;

FIG. 4A is a graph illustrating an example of output data based on anintensity value before correction processing, and FIG. 4B is a graphillustrating an example of output data based on an intensity value aftercorrection processing, with the fine particle measuring apparatus.

FIG. 5 is a graph illustrating a result that a detection wavelength ofeach PMT of a PMT array is determined by a flow cytometer which isexperimentally produced in the embodiment;

FIG. 6 is a graph illustrating a result that a relative sensitivity ofeach PMT of the PMT array is calculated by the flow cytometer which isexperimentally produced in the embodiment;

FIGS. 7A to 7D are graphs illustrating spectrum charts of fluorescencebeads obtained by measurement with a spectrophotofluorometer in theembodiment;

FIGS. 8A to 8C are graphs illustrating spectrum charts of fluorescencebeads FPK505 obtained by measurement with the flow cytometer which isexperimentally produced in the embodiment, in which FIG. 8A illustratesa chart before the correction processing, FIG. 8B illustrates a chartbased on a first corrected intensity value, and FIG. 8C illustrates achart based on a second corrected intensity value;

FIGS. 9A to 9C are graphs illustrating spectrum charts of fluorescencebeads FPK505 obtained by measurement with the flow cytometer which isexperimentally produced in the embodiment, in which FIG. 9A illustratesa chart before the correction processing, FIG. 9B illustrates a chartbased on a first corrected intensity value, and FIG. 9C illustrates achart based on a second corrected intensity value;

FIGS. 10A to 10C are graphs illustrating spectrum charts of fluorescencebeads FPK528 obtained by measurement with the flow cytometer which isexperimentally produced in the embodiment, in which FIG. 10A illustratesa chart before the correction processing, FIG. 10B illustrates a chartbased on a first corrected intensity value, and FIG. 10C illustrates achart based on a second corrected intensity value;

FIGS. 11A to 11C are graphs illustrating spectrum charts of fluorescencebeads FPK549 obtained by measurement with the flow cytometer which isexperimentally produced in the embodiment, in which FIG. 11A illustratesa chart before the correction processing, FIG. 11B illustrates a chartbased on a first corrected intensity value, and FIG. 11C illustrates achart based on a second corrected intensity value; and

FIGS. 12A to 12C are graphs illustrating spectrum charts of fluorescencebeads FPK667 obtained by measurement with the flow cytometer which isexperimentally produced in the embodiment, in which FIG. 12A illustratesa chart before the correction processing, FIG. 12B illustrates a chartbased on a first corrected intensity value, and FIG. 12C illustrates achart based on a second corrected intensity value.

DETAILED DESCRIPTION

Preferred embodiments of the present application will be described belowin reference to the accompanying drawings. It should be noted thatembodiments described below are merely an example of a typicalembodiment of the present application and the scope of the presentapplication is not interpreted limitedly by this example. Thedescription will be given in the following order.

1. Fine Particle Measuring Apparatus According to an Embodiment

(1) Configuration of Apparatus

(2) Correction Processing of Fluorescence Intensity

-   -   [Calculation of First Corrected Intensity Value]    -   [Calculation of Second Corrected Intensity Value]

(3) Data Display

(4) Data Analysis

2. Fine Particle Measuring Apparatus According to Another Embodiment

(1) Configuration of Apparatus

(2) Correction Processing of Fluorescence Intensity

-   -   [Calculation of First Corrected Intensity Value]    -   [Calculation of Second Corrected Intensity Value]

(3) Data Display

1. Fine Particle Measuring Apparatus According to an Embodiment

(1) Configuration of Apparatus

FIG. 1 is a block diagram illustrating the functional configuration of afine particle measuring apparatus A according to an embodiment of thepresent application. FIG. 2 schematically illustrates the configurationof a detection unit 10 of the fine particle measuring apparatus A.

The fine particle measuring apparatus A includes the detection unit 10which detects fluorescence emitted from fine particles by irradiatingthe fine particles with laser light and converts the intensity of thedetected fluorescence into an electric signal to output the electricsignal, a CPU 20, a memory 30, and a hard disk (storage unit) 40. In thefine particle measuring apparatus A, the CPU 20, the memory 30, and thehard disk (storage unit) 40 constitute a processing unit. The fineparticle measuring apparatus A further includes a mouse 51, a keyboard52, and a display unit 60 which is composed of a display 61 and aprinter 62, as a user interface.

The detection unit 10 may have the configuration similar to that of arelated art fine particle measuring apparatus. In particular, thedetection unit 10 is composed of an irradiation system which condenseslaser light from a light source 101 and irradiates fine particles P withthe laser light and a detection system which includes a spectral element102 which divides fluorescence emitted from fine particles P and alight-receiving element array 103 which detects the divided light. Inthe fine particle measuring apparatus A, fine particles P flow inside aflow path, which is formed in a flow cell or a microchip, in a manner tobe arranged in a line.

The irradiation system includes a condenser lens for condensing laserlight and irradiating fine particles P with the laser light, a dichroicmirror, a band pass filter, and the like (not depicted), other than thelight source 101. Here, the light source 101 may be a light sourceobtained by combining two or more light sources which emit light beamshaving different wavelengths from each other. In this case, spots offine particles P which are irradiated with two or more types of laserlight may be same as each other or different from each other. Further,the detection system may include a condenser lens (not depicted) whichcondenses fluorescence emitted from fine particles P and introduces thefluorescence to the spectral element 102, for example. In this example,the configuration which employs a photo multiplier tube (PMT) array inwhich PMTs of 32 channels of different detection wavelength bands areone-dimensionally arranged is described as the light-receiving elementarray 103. Here, as the light-receiving element array 103, a photodiodearray or an array in which a plurality of independent detection channelsof different detection wavelength bands such as two-dimensionallight-receiving elements which are CCDs or CMOSs are arranged may beemployed.

In the fine particle measuring apparatus A, the detection unit 10 may beconfigured to detect not only fluorescence but also light which isemitted from fine particles P by irradiation of laser light, i.e.scattering light such as forward-scattering light, side-scatteringlight, Rayleigh scattering, and Mie scattering, and the like. Moreover,it should be reasonably understood that the light-receiving elementarray 103 may detect the frequency range of the divided light.

(2) Correction Processing of Fluorescence Intensity

The CPU 20 and the memory 30 perform correction processing of anintensity value of fluorescence based on an electric signal outputtedform the detection unit 10 in collaboration with a fluorescenceintensity correcting program 41 and an OS 43 which are stored in thehard disk 40. This correction processing includes a process ofcorrecting an intensity value of fluorescence by a detection wavelengthbandwidth of each light-receiving element (in this example, PMTs ofchannels 1 to 32) so as to calculate a first corrected intensity valueand a process of correcting the first corrected intensity value by usingsensitive data of each PMT so as to calculate a second correctedintensity value. It is also reasonably contemplated that the correctedintensity value may be based on a frequency range as an alternative tothe wavelength bandwidth due to the inversely proportional relationshipbetween frequency and the wavelength of light.

[Calculation of First Corrected Intensity Value]

Calculation of a first corrected intensity value is performed bydividing an intensity value of fluorescence acquired in each PMT by adetection wavelength bandwidth of a corresponding PMT.

In particular, the n-th intensity value obtained at a PMT of channel kamong PMTs of channels 1 to 32 is denoted as I[k,n], a detection lowerlimit wavelength of the PMT of the channel k is denoted as L[k], and adetection upper limit wavelength is denoted as H[k]. In this case, afirst corrected intensity value J₁[k,n] is calculated by the followingexpression. Here, k denotes an integer from 1 to 32.

J ₁ [k,n]=I[k,n]/(H[k]−L[k])

When the optical system of the detection unit 10 including the spectralelement 102 has nonlinearity, wavelength bandwidths of light beams whichare detected at PMTs of the channels 1 to 32 are different from eachother among the PMTs (refer to FIG. 5 described later). Therefore,intensity values of fluorescence acquired in the respective PMTs arerelatively large in channels of which detection wavelength width islarge and are relatively small in channels of which detection wavelengthwidth is small, causing a measurement error.

Especially, in a case where fluorescence divided by the spectral element102 is detected with the light-receiving element array 103 so as tomeasure a fluorescence spectrum of fine particles P, distortion of thespectrum shape is caused by the above-described measurement error in thefluorescence spectrum in which intensity values of fluorescence acquiredin respective PMTs are directly used. That is, when a two-dimensionalgraph (referred to below as a “spectrum chart”) of which the horizontalaxis expresses a channel number and a vertical axis expresses anintensity value is compared to a spectrum chart of which a horizontalaxis expresses a detection wavelength and a vertical axis expresses anintensity value, the intensity value is relatively larger in channels ofwhich a detection wavelength width is larger, in the former chart.Therefore, fluorescence spectrum shapes of the both charts do not accordwith each other. Thus, there is a gap between the fluorescence spectrumshapes.

With the first corrected intensity value which is obtained by dividingan intensity value of fluorescence acquired in each PMT by a detectionwavelength bandwidth of a corresponding PMT, a measurement error causedby such nonlinearity of an optical system can be compensated.

The detection wavelength widths (H[k]−L[k]) of the respective PMTs areuniquely determined depending on a type and an arrangement of opticalelements such as the spectral element 102, a condenser lens, a dichroicmirror, and a bandpass filter, which constitute the detection unit 10(refer to FIG. 5 described later). Accordingly, if detection wavelengthwidths of respective PMTs are acquired at a stage on which apparatusdesign including selection and arrangement of optical elements iscompleted, a first corrected intensity value can be calculated based onan intensity value of fluorescence acquired at each of the PMTs.

[Calculation of Second Corrected Intensity Value]

Calculation of a second corrected intensity value is performed bydividing the first corrected intensity value acquired in each PMT by arelative sensitivity of a corresponding PMT.

In particular, a relative sensitivity of the PMT of the channel k amongthe PMTs of the channels 1 to 32 is denoted as S[k]. In this case, asecond corrected intensity value J₂[k,n] is calculated from thefollowing expression.

J ₂ [k,n]=J ₁ [k,n]/S[k]

Here, the relative sensitivity is obtained such that an intensity valueobtained in each channel by irradiating the PMT with light beams havingthe same intensities and wavelengths is expressed by a relative valuewith respect to an intensity value of a channel at which the largestintensity value is obtained. The relative sensitivity can bepreliminarily calculated from sensitivity data in which electric signalamounts, which are outputted from respective channels when the PMTs areirradiated with light beams having the same intensities and wavelengths,are recorded. In this sensitivity data, sensitivity difference existingin respective PMTs and sensitivity difference (gain) which is set inrespective PMTs by a user are both reflected. Here, the gain can bearbitrarily adjusted by changing a setting value such as an appliedvoltage by a user.

Sensitivities of the PMTs of the channels 1 to 32 are different amongthe PMTs due to individual difference of the PMTs and setting differenceof the gain (refer to FIG. 6 described later). Therefore, the intensityvalues of fluorescence acquired in respective PMTs are relatively largein channels of which the sensitivity is high and are relatively small inchannels of which the sensitivity is low, also causing a measurementerror.

Especially, in a case where fluorescence divided by the spectral element102 is detected with the light-receiving element array 103 so as tomeasure a fluorescence spectrum of fine particles P, distortion of thespectrum shape is caused by the above-described measurement error in thefluorescence spectrum in which intensity values of fluorescence acquiredin respective PMTs are directly used. That is, the intensity value isrelatively larger in a channel of which the sensitivity is higher, sothat the shape of the fluorescence spectrum is distorted. Thus, thefluorescence spectrum is not accurate.

With the second corrected intensity value which is obtained by dividingthe first corrected intensity value of each PMT by a relativesensitivity of a corresponding PMT, a measurement error caused by suchsensitivity difference among light-receiving elements can becompensated.

(3) Data Display

The processing unit which includes the CPU 20, the memory 30, and thehard disk 40 forms a spectrum chart having a coordinate axis of thefirst corrected intensity value or the second corrected intensity valuewhich is calculated, so as to output the spectrum chart to the displayunit 60.

The spectrum chart may be formed such that the horizontal axis expressesa detection wavelength of each PMT and the vertical axis expresses thefirst corrected intensity value (refer to FIGS. 8B, 9B, 10B, 11B, and12B described later). Further, the spectrum chart is preferably formedsuch that the horizontal axis expresses a detection wavelength and thevertical axis expresses the second corrected intensity value (refer toFIGS. 8C, 9C, 10C, 11C, and 12C described later).

In the spectrum chart, an intensity value may be expressed by astatistical number such as an average value, a standard error, a mediumvalue, and a quartile point depending on the number of fine particles(number of events or density) which are detected based on apredetermined fluorescence intensity value in a predetermined detectionwavelength (refer to FIGS. 9A to 9C described later). Further, thespectrum chart can be displayed as a three-dimensional graph to which acoordinate axis expressing the number of events is added and thisthree-dimensional graph can be pseudo-3D-displayed. Further, thespectrum chart can be multicolor-displayed by hue and saturation and/orlightness to which information (frequency information) about the numberof fine particles (number of events or density) is reflected (refer toFIGS. 8A to 8C described later).

(4) Data Analysis

The processing unit which includes the CPU 20, the memory 30, and thehard disk 40 can generate spectrum data using the first correctedintensity value or the second corrected intensity value, which iscalculated, as a first parameter and a detection wavelength of each PMTas a second parameter so as to execute various kinds of analysis byusing the spectrum data. Here, the first parameter may be a statisticalnumber such as an average value of first corrected intensity values orsecond corrected intensity values, a standard error, a medium value, anda quartile point, which are obtained by calculating all or part of aplurality of fine particles P which are measured.

Further, the processing unit can compare the generated spectrum datawith reference spectrum data stored in the hard disk (storage unit) 40so as to evaluate a degree of accordance between these pieces of data.Further, the processing unit can output the evaluation result to thedisplay unit 60. Here, the reference spectrum data may be eitherspectrum data which is obtained such that fine particles including apreviously-identified fluorescence substance are preliminarily measuredby the fine particle measuring apparatus A and the above-describedcorrection processing is performed, or spectrum data which is obtainedby measuring a fluorescence spectrum of the fluorescence substance by acommon spectrophotofluorometer. This is because a measurement valueobtained by the fine particle measuring apparatus A can be directlycompared to a measurement value obtained by a commonspectrophotofluorometer due to correction. The reference spectrum datais stored in the hard disk 40 as reference data 42. As the evaluationmethod of the degree of accordance with the reference spectrum data, asum of difference in respective detection wavelengths, a sum of absolutevalues of difference, or a square sum of difference, for example, can beused.

If the spectrum data which is obtained by measuring fine particles P bythe fine particle measuring apparatus A and correcting the measurementresult is compared with a piece or two or more pieces of referencespectrum data, whether fluorescence emitted from the measured fineparticles P is similar to any of fluorescence recorded in the referencespectrum data can be determined. Accordingly, in a case where a kind ofa fluorescence substance included in the measured fine particles P isunclear, for example, a fluorescence substance having high degree ofsimilarity can be searched from the recorded reference spectrum data soas to predict a kind of a fluorescence substance included in the fineparticles P.

Further, if the spectrum data which is obtained by measuring fineparticles P by the fine particle measuring apparatus A and correctingthe measurement result is compared with reference spectrum data which ispreliminarily obtained by measuring the same fine particles P, the stateof the fine particle measuring apparatus A can be evaluated. That is,there is a case where the state of the fine particle measuring apparatusA is deteriorated due to an effect of operation abnormality of thelight-receiving element array, turbulence of flow of fine particles in aflow cell or a microchip, a gap due to temperature change or vibrationof each element such as a lens and a spectral element, and the like andtherefore, accuracy of measurement is degraded. It can be consideredthat measuring identical samples by the fine particle measuringapparatus A and the spectrophotofluorometer and comparing the results tocheck such apparatus state is an effective way. According to theembodiment of the present application, a measurement result obtained bythe fine particle measuring apparatus A can be directly compared to ameasurement result of the spectrophotofluorometer, so that the state canbe simply and accurately evaluated.

In addition, if compensation processing using a plurality of pieces ofreference spectrum data is performed with respect to spectrum data whichis obtained by measuring fine particles P by the fine particle measuringapparatus A and correcting the measurement result, quantity of aplurality of fluorescence coloring matters included in fine particles Pcan be determined. For example, when fine particles P are dyed by aplurality of coloring matters {D₁, D₂, . . . , D_(n)}, it is preferablethat fluorescence coloring matters obtained from samples which aresingly dyed by respective fluorescence coloring matters {D₁, D₂, . . . ,D_(n)} be included in reference spectrum data used in the compensationprocessing. As the method of the compensation processing, a least-squaremethod may be employed, for example.

2. Fine Particle Measuring Apparatus According to Another Embodiment

(1) Configuration of Apparatus

FIG. 3 schematically illustrates the configuration of a detection unit10 of a fine particle measuring apparatus B according to anotherembodiment of the present application. The functional configuration ofthe fine particle measuring apparatus B is same as that of the fineparticle measuring apparatus A, which is depicted in FIG. 1, accordingto the embodiment described above, so that the description thereof isskipped.

The fine particle measuring apparatus B is different from the fineparticle measuring apparatus A which measures intensities of light beamsin continuous wavelength bands as a fluorescence spectrum, on the pointthat the fine particle measuring apparatus B selects a plurality oflight beams in discontinuous wavelength bands by using a wavelengthselecting element such as a filter and measures intensities of the lightbeams of respective wavelength bands.

The detection unit 10 of the fine particle measuring apparatus B iscomposed of an irradiation system which condenses laser light from alight source 101 and irradiates fine particles P with the laser lightand a detection system which includes wavelength selecting elements 104to 106 which select light in a predetermined wavelength band fromfluorescence emitted from fine particles P and light-receiving elements107 to 110 which detect the selected light. In the fine particlemeasuring apparatus B, fine particles P flow inside a flow path, whichis formed in a flow cell or a microchip, in a manner to be arranged in aline.

The irradiation system includes a condenser lens for condensing laserlight and irradiating fine particles P with the laser light, a dichroicmirror, a band pass filter, and the like (not depicted), other than thelight source 101. Here, the light source 101 may be a light sourceobtained by combining two or more light sources which emit light beamshaving different wavelengths from each other. In this case, spots offine particles P which are irradiated with two or more types of laserlight may be same as each other or different from each other. Further,the detection system may include a condenser lens (not depicted) whichcondenses fluorescence emitted from fine particles P and introduces thefluorescence to the wavelength selecting element 104, for example. Inthis example, the configuration which employs photo multiplier tubes(PMTs) of different detection wavelength bands as the light-receivingelements 107 to 110 is described. Hereinafter, the channel numbers ofthe light-receiving elements 107 to 110 are respectively 1 to 4. Here,as the light-receiving elements 107 to 110, photodiodes may be used.

In the fine particle measuring apparatus B, the detection unit 10 can beconfigured to detect scattering light and the like as well asfluorescence, as is the case with the fine particle measuring apparatusA.

(2) Correction Processing of Fluorescence Intensity

The fine particle measuring apparatus B performs correction processingof an intensity value of fluorescence based on an electric signaloutputted from the detection unit 10. This correction processingincludes a process of correcting an intensity value of fluorescence by adetection wavelength bandwidth of each light-receiving element (in thisexample, PMTs of channels 1 to 4) so as to calculate a first correctedintensity value and a process of correcting the first correctedintensity value by using sensitive data of each PMT so as to calculate asecond corrected intensity value.

[Calculation of First Corrected Intensity Value]

Calculation of a first corrected intensity value is performed bydividing an intensity value of fluorescence acquired in each PMT by adetection wavelength bandwidth of a corresponding PMT.

In particular, the n-th intensity value obtained at a PMT of channel kamong PMTs of channels 1 to 4 is denoted as I[k,n], a detection lowerlimit wavelength of the PMT of the channel k is denoted as L[k], and adetection upper limit wavelength is denoted as H[k]. In this case, afirst corrected intensity value J₁[k,n] is calculated by the followingexpression. Here, k denotes an integer from 1 to 4.

J ₁ [k,n]=I[k,n]/(H[k]−L[k])

When the optical system of the detection unit 10 including thewavelength selecting elements 104 to 106 has nonlinearity, wavelengthbandwidths of light beams which are detected at PMTs of the channels 1to 4 are different from each other among the PMTs. Therefore, intensityvalues of fluorescence acquired in the respective PMTs are relativelylarge in channels of which detection wavelength width is large and arerelatively small in channels of which detection wavelength width issmall, causing a measurement error.

With the first corrected intensity value which is obtained by dividingan intensity value of fluorescence acquired in each PMT by a detectionwavelength bandwidth of a corresponding PMT, a measurement error causedby such nonlinearity of an optical system can be compensated.

The detection wavelength widths (H[k]−L[k]) of the respective PMTs areuniquely determined depending on a type and an arrangement of opticalelements such as the wavelength selecting elements 104 to 106, acondenser lens, a dichroic mirror, and a bandpass filter, whichconstitute the detection unit 10. Accordingly, if detection wavelengthwidths of respective PMTs are acquired at a stage on which apparatusdesign including selection and arrangement of optical elements iscompleted, a first corrected intensity value can be calculated based onan intensity value of fluorescence acquired at each of the PMTs.

[Calculation of Second Corrected Intensity Value]

Calculation of a second corrected intensity value is performed bydividing the first corrected intensity value acquired in each PMT by arelative sensitivity of a corresponding PMT.

In particular, a relative sensitivity of the PMT of the channel k amongthe PMTs of the channel 1 to 4 is denoted as S[k]. In this case, asecond corrected intensity value J₂[k,n] is calculated from thefollowing expression.

J ₂ [k,n]=J ₁ [k,n]/S[k]

Sensitivities of the PMTs of the channels 1 to 4 are different among thePMTs due to individual difference of the PMTs and setting difference ofthe gain. Therefore, the intensity values of fluorescence acquired inrespective PMTs are relatively large in channels of which thesensitivity is high and are relatively small in channels of which thesensitivity is low, also causing a measurement error.

With the second corrected intensity value which is obtained by dividingthe first corrected intensity value of each PMT by a relativesensitivity of corresponding PMT, a measurement error caused by suchsensitivity difference among light-receiving elements can becompensated.

(3) Data Display

The fine particle measuring apparatus B forms a two-dimensional graphhaving a coordinate axis of the first corrected intensity value or thesecond corrected intensity value which is calculated, and outputs thetwo-dimensional graph to the display unit 60.

The two-dimensional graph may be formed such that the horizontal axisexpresses a detection wavelength of each PMT and the vertical axisexpresses the first corrected intensity value or the second correctedintensity value. FIG. 4A illustrates a graph before the correctionprocessing, and FIG. 4B illustrates a graph based on the secondcorrected intensity value. FIG. 4A is a graph of which the horizontalaxis expresses the channel number k (k is an integer from 1 to 4) andthe vertical axis expresses a logarithm of an intensity value (I[k]) offluorescence acquired in each channel. Further, FIG. 4B is a graph ofwhich the horizontal axis expresses a detection wavelength and thevertical axis expresses a logarithm of a corrected value (J₁ [k] orJ₂[k]) of a fluorescence intensity value acquired in each channel.

By the above-described correction processing, the fine particlemeasuring apparatus B can display a measurement result of intensities oflight in discontinuous wavelength bands which are selected by using thewavelength selecting element, in a state that a measurement error causedby nonlinearity of the optical system of the apparatus and sensitivitydifference among the light-receiving elements is compensated.

The fine particle measuring apparatus according to the embodiments ofthe present application may have the following configuration.

In an embodiment, a fine particle measuring apparatus includes adetection unit configured to detect light emitted from a fine particle,and a processing unit having a memory device storing instructions whichwhen executed by the processing unit causes the processing unit tocalculate a corrected intensity value of the detected light, andgenerate spectrum data based on the corrected intensity value.

In the fine particle measuring apparatus, the detection unit may beconfigured to generate an intensity value of the detected light.

In the fine particle measuring apparatus, the detection unit may includea plurality of light-receiving elements.

In the fine particle measuring apparatus, the processing unit maycalculate the corrected intensity value based on a detection wavelengthbandwidth of each of the plurality of light-receiving elements.

In the fine particle measuring apparatus, the processing unit maycalculate the corrected intensity value based on a detection frequencyrange of each of the plurality of light-receiving elements.

In the fine particle measuring apparatus, the processing unit maycalculate the corrected intensity value based on at least one of adetection wavelength bandwidth and a detection frequency range of eachof the plurality of light-receiving elements.

In the fine particle measuring apparatus, the corrected intensity valuemay be a first corrected intensity value and the processing unit furthercalculates a second corrected intensity value based on the firstcorrected intensity value and relative sensitivity data of eachcorresponding one of the plurality of light-receiving elements.

In the fine particle measuring apparatus, the memory device storinginstructions which when executed by the processor may cause theprocessor to compare the spectrum data with reference spectrum data.

The fine particle measuring apparatus may further include a display, andthe memory device storing instructions which when executed by theprocessor causes the processor to display results of the comparison ofspectrum data with the reference spectrum data.

In another embodiment, a device for receiving data from a detection unitis provided. The detection unit has a plurality of light-receivingelements configured to detect light emitted from a fine particle andconvert the detected light to a corresponding intensity value. Thedevice includes a processor and a memory device storing instructionswhich when executed by the processor, cause the processor to receive theintensity value of the detected light, correct the intensity value, andgenerate spectrum data based on the corrected intensity value.

In the device, the processor may correct the intensity value based on atleast one of a detection wavelength bandwidth and a detection frequencyrange of each corresponding one of the plurality of light-receivingelements.

In the device, the memory device storing instructions which whenexecuted by the processor may cause the processor to compare thespectrum data with reference spectrum data.

The device may further include a display, and the memory device storinginstructions which when executed by the processor, cause the processorto display results of the comparison of spectrum data with the referencespectrum data.

In another embodiment, a device for receiving a corrected intensityvalue of detected light emitted from a fine particle includes aprocessor, and a memory device storing instructions which when executedby the processor, cause the processor to receive the corrected intensityvalue of the detected light, and generate spectrum data based on thecorrected intensity value.

In the device, the memory device storing instructions which whenexecuted by the processor, may cause the processor to compare thespectrum data with reference spectrum data.

The device may further include a display, and the memory device storinginstructions which when executed by the processor, cause the processorto display results of the comparison of spectrum data with the referencespectrum data.

In another embodiment, a method for analyzing data includes detectinglight emitted from a fine particle, correcting an intensity value of thedetected light, and generating spectrum data based on the correctedintensity value.

In the method for analyzing data, the step of correcting the intensityvalue of detected light is may be based on at least one of a detectionwavelength bandwidth and a detection frequency range corresponding tothe detected light.

Example

A spectrum type flow cytometer including the detection unit illustratedin FIG. 2 was produced experimentally. As a light source, a laser diodeof the wavelength of 488 nm and a laser diode of the wavelength of 638nm were used. Further, as a spectral element, a prism array obtained bycombining a plurality of prisms was used. As a light-receiving elementarray, a PMT array of 32 channels was used and fluorescence of thewavelength from 500 nm to 800 nm was divide-detected.

FIG. 5 illustrates a graph of a result that detection wavelength bandsof respective PMTs are determined in the trial apparatus. “x” in thegraph denotes a detection lower limit wavelength (L[k]) of a PMT of eachchannel and “◯” denotes a detection upper limit wavelength (H[k]). Here,k denotes an integer from 1 to 32. It can be confirmed that a detectionwavelength bandwidth of a PMT in a long-wavelength side at which thechannel number is large is larger among detection wavelength bandwidths(H[k]−L[k]) of respective PMTs. Here, in PMTs around the channel 21stwhich detect fluorescence of the wavelength around 638 nm, detectedfluorescence is also restricted by an optical filter which prevents leakof laser light from a light source of the wavelength of 638 nm.

FIG. 6 illustrates a graph of calculation results of relativesensitivities of respective PMTs. The relative sensitivity is obtainedsuch that intensity values which are obtained in respective channels byirradiating respective PMTs with light beams having the same intensitiesand wavelengths are expressed by relative values with respect to anintensity value of the channel 32, at which the strongest intensityvalue could be obtained, in a manner that the intensity value of thechannel 32 is set to 1.

First, fluorescence spectrums of commercially-available fluorescencebeads were measured by using an F-4500 type spectrophotofluorometer(Hitachi High-Technologies Corporation). As the fluorescence beads, fourkinds of beads which were fluorescent particle kit (FPK) 505, FPK528,FPK549, and FPK667 obtained from Spherotech, Inc. were used. Obtainedspectrum charts (reference spectrum charts) are illustrated in FIGS. 7Ato 7D. FIG. 7A, FIG. 7B, FIG. 7C, and FIG. 7D respectively illustratefluorescence spectrums of FPK505, FPK528, FPK549, and FPK667. Thehorizontal axes express a fluorescence wavelength (500 nm to 800 nm) andthe vertical axes express a fluorescence intensity value (indicated inlogarithm). Here, an excitation wavelength of the laser light is thewavelength of 488 nm in FIGS. 7A to 7C and is the wavelength of 638 nmin FIG. 7D.

Fluorescence spectrums of the fluorescence beads were subsequentlymeasured by using the trial apparatus. The obtained spectrum charts areillustrated in FIGS. 8A to 12C. FIGS. 8A to 9C illustrate charts of theFPK505, FIGS. 10A to 10C illustrate charts of the FPK528, FIGS. 11A to11C illustrate charts of the FPK549, and FIGS. 12A to 12C illustratecharts of the FPK667. In FIGS. 8A to 8C, the number of events ofrespective channels is displayed by colors of the spectrums. Further, inFIGS. 9A to 9C, an intensity value is expressed by an average value(solid line) based on the number of events and an average value±astandard deviation (dashed line).

FIGS. 8A, 9A, 10A, 11A, and 12A are spectrum charts of which thehorizontal axis expresses the channel number and the vertical axisexpresses a logarithm of an intensity value (I[k], k denotes an integerfrom 1 to 32) of fluorescence obtained in each channel.

Spectrum shapes depicted in the spectrum charts of FIGS. 8A, 9A, 10A,11A, and 12A are obviously different from spectrum shapes of thereference spectrum charts which are depicted in FIGS. 7A to 7D. Thisrepresents that the spectrum shape is distorted due to a measurementerror caused by nonlinearity of an optical system of the apparatus andsensitivity difference among light-receiving elements, in a fluorescencespectrum obtained by directly using an intensity value (I[k]) offluorescence obtained at a PMT.

FIGS. 8B, 9B, 10B, 11B, and 12B are spectrum charts of which thehorizontal axis expresses a detection wavelength and the vertical axisexpresses an logarithm of an first corrected value (J₁ [k], k denotes aninteger from 1 to 32) of a fluorescence intensity value which isobtained in each channel. The first corrected intensity value J₁[k] wasobtained by dividing the intensity value (I[k]) of fluorescence obtainedin each PMT by a detection wavelength bandwidth (H[k]−L[k]), which isdepicted in FIG. 5, of a corresponding PMT. More specifically, the n-thintensity value I[k,n] obtained at the PMT of the channel k was dividedby a detection wavelength bandwidth (H[k]−L[k]) of the PMT to obtain thefirst corrected intensity value J₁[k,n], and distribution of J₁[k,n] wasdrawn in a range L[k] to H[k] of the horizontal axis so as to form aspectrum chart.

The spectrum shapes depicted in the spectrum charts of FIGS. 8B, 9B,10B, 11B, and 12B are approximately accorded with the spectrum shapes ofthe reference spectrum charts of FIGS. 7A to 7D. This represents that ameasurement error caused by nonlinearity of the optical system of theapparatus was compensated by the correction processing in which anintensity value (I[k]) of fluorescence obtained in each PMT was dividedby a detection wavelength bandwidth (H[k]−L[k]) of a corresponding PMT,and distortion of the spectrum shapes could be corrected.

FIGS. 8C, 9C, 10C, 11C, and 12C are spectrum charts of which thehorizontal axis expresses a detection wavelength and the vertical axisexpresses an logarithm of an second corrected value (J₂[k], k denotes aninteger from 1 to 32) of a fluorescence intensity value which isobtained in each channel. The second corrected intensity value J₂[k] wasobtained by dividing the first corrected intensity value (J₁[k]) by arelative sensitivity (S[k]), which is depicted in FIG. 6, of acorresponding PMT.

Spectrum shapes depicted in spectrum charts of FIGS. 8C, 9C, 10C, 11C,and 12C are well accorded with spectrum shapes of the reference spectrumcharts depicted in FIGS. 7A to 7D. Especially, though distortion of thespectrum shapes which seems to be caused by sensitive difference of PMTsis observed in a region of the wavelength of about 500 nm in thespectrum charts based on the first corrected value (J₁[k]) of FIGS. 8B,9B, 10B, 11B, and 12B, this distortion is corrected in the spectrumcharts based on the second corrected value (J₂[k]) of FIGS. 8C, 9C, 10C,11C, and 12C. This has proved that a measurement error caused bysensitivity difference among light-receiving elements is compensated bythe correction processing in which the first corrected value (J₁[k]) isdivided by a relative sensitivity (S[k]) of a corresponding PMT anddistortion of the spectrum shapes can be corrected.

According to the fine particle measuring apparatus of the embodiments ofthe present application, from the above-described results, it can beunderstood that a spectrum shape, which is well accorded with areference spectrum chart which is obtained by measuring by a commonspectrophotofluorometer, of fine particles can be measured.

Therefore, in the fine particle measuring apparatus according to theembodiments of the present application, a spectrum shape of a sample ofwhich the type or the quantity of marking fluorescence coloring mattersare not previously identified is detected from a database (referencedata) in which a spectrum shape of fluorescence which is previouslyidentified is recorded, being able to predict the type or the quantityof the fluorescence coloring matters marked on the sample.

Further, in the fine particle measuring apparatus according to theembodiments of the present application, compensation calculationperformed when a sample marked by a plurality of fluorescence coloringmatters is analyzed can be performed by using a fluorescence spectrum ofeach fluorescence coloring matter which is preliminarily measuredwithout performing measurement, which has been performed in related art,of a sample which is singly dyed by each fluorescence coloring matter.Accordingly, work and time for sample analysis and resources of reagentsand the like can be reduced.

It should be understood that various changes and modifications to thepresently preferred embodiments described herein will be apparent tothose skilled in the art. Such changes and modifications can be madewithout departing from the spirit and scope of the present subjectmatter and without diminishing its intended advantages. It is thereforeintended that such changes and modifications be covered by the appendedclaims.

The invention is claimed as follows:
 1. A fine particle measuringapparatus, comprising: a detection unit configured to detect light froma fine particle by a plurality of light-receiving elements of differentdetection wavelength bands; and a processing unit configured to correctan intensity value of the light obtained by the detection unit by adetection wavelength bandwidth of each of the light-receiving elementsso as to calculate a first corrected intensity value.
 2. The fineparticle measuring apparatus according to claim 1, wherein the detectionunit includes a spectral element configured to divide light from thefine particle and a light-receiving element array in which a pluralityof light-receiving elements of different detection wavelength bands arearranged.
 3. The fine particle measuring apparatus according to claim 1,wherein the processing unit forms a spectrum chart in which an axisexpresses the detection wavelength and another axis expresses the firstcorrected intensity value, so as to output the spectrum chart to adisplay unit.
 4. The fine particle measuring apparatus according toclaim 1, wherein the processing unit generates spectrum data using thedetection wavelength as a parameter and the first corrected intensityvalue as another parameter, and compares the spectrum data withreference spectrum data that is stored in a storage unit, so as tooutput whether the both data are accorded with each other or are notaccorded with each other to the display unit.
 5. The fine particlemeasuring apparatus according to claim 1, wherein the processing unitcorrects the first corrected intensity value by using sensitivity dataof each of the light-receiving elements so as to calculate a secondcorrected intensity value.
 6. The fine particle measuring apparatusaccording to claim 5, wherein the processing unit forms a spectrum chartin which an axis expresses the detection wavelength and another axisexpresses the first corrected intensity value or the second correctedintensity value, so as to output the spectrum chart to a display unit.7. The fine particle measuring apparatus according to claim 5, whereinthe processing unit generates spectrum data using the detectionwavelength as a parameter and the first corrected intensity value or thesecond corrected intensity value as another parameter, and compares thespectrum data with reference spectrum data that is stored in a storageunit, so as to output whether the both data are accorded with each otheror are not accorded with each other to the display unit.
 8. The fineparticle measuring apparatus according to claim 1 that is a spectrumtype flow cytometer.